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Патент USA US3087873

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United States Patent
3,087,864
Patented Apr. 30, 1963
1
2
3,087,864
buifer pH 7.3. While a pH range of S to 10 is usually
permissible, a pH range of from 6 to 8 has been found
ideal. The desired steroid is added and after comple
PROCESS FOR THE 1,2-HYDROGENATION 0F
STEROIDS BY THE USE OF DRIED THALLI
Louis I. Feldman, Spring Valiey, and Chester E. Holm
lund, Pearl River, N.Y., and Norma L. Barbacci, Mout
tion of the 1,2-dehydrogenation, the reaction mixture is
extracted with the usual water immiscible steroid extract
ing solvents. Puri?cation is carried out employing the
usual puri?cation techniques well known to those skilled
in the art. The desired LZ-dehydrogcnated steroid is
vale, N.J., assignors to American Cyanamid Company,
New York, N.Y., a corporation of Maine
No Drawing. Filed Apr. 28, 1961, Ser. No. 106,150
19 Claims. (Cl. 195-51)
obtained by direct crystallization or, in some cases,
10 chromatography can be used to advantage.
This invention relates to a new method of biologically
The use of dried thalli to carry out the reaction which
dehydrogenating steroids.
More particularly, it relates
is divorced from the growth phase of the organism has
many advantages. ‘The enzymatic reaction is the desired
objective, and the growing of the organism which is the
to the 1,2-dehydrogenation of steroids by a new process.
‘In the past it has been mentioned that preparations
other than growing cells could be used for steroid trans
formations, however, transformations in the literature are
source of many undesirable features such as variability,
high dilution, extraneous material, necessity of steriliza
almost invariably carried out using growing cells. The
outstanding utility of preparations used for steroid trans—
tion and asepsis is eliminated, numerous advantages are
shown by the dry thalli preparations. One of the prin
formations which are free of growing cells has not been
cipal advantages is a standard source of enzyme from
20 which standard 1,2-dchydrogenation conditions can be
demonstrated.
The new process of the present invention comprises
derived providing a higher degree of control and pre
preparing and using a dry powder for the various reac
dictability than can be obtained with growing cells. A
further advantage of the dry thalli preparation is the use
tions, such as, dehydrogenation of steroids, which is pre
pared by harvesting microorganisms, separating the super
of higher steroid concentrations in the transformation
natant liquid by ?ltration or centrifugation, precipitating
reaction thus reducing to a minimum the unfortunate
the thalli in acetone and filtering the acetone treated
dilution factor inherent in using growing cells for fermen
thalli followed by air drying at room temperature or
tation. A still further advantage of the dry thalli is
alternatively lyophilizing the thalli after separation from
that it permits through the removal of extraneous mash
the supernatant beer, and washing. The term “thalli” is
material higher isolation yields, simpler isolation pro
used to describe the cell body obtained after, for example, 30 cedures and a more accurate assay of the desired ?nal
centrifugation and also includes mycelia. The thalli pow
product. Also, use of the dried thalli is advantageous as
der so prepared has shown remarkable activity for more
a time-saving factor since the absence of a growth phase
at the time of 1,2-dehydrogenation and the lack of neces
than a year and, furthermore, after exposure to the
steroid desired to be dehydrogenated may be reused in
one or more consecutive new transformations.
sity of sterilization and aseptic techniques save consid
erable time. The dried thalli preparation is more stable
than growing cells and the thalli may be recovered after
completion of a 1,2-dehydrogenation reaction and be used
It also
appears that identical thalli preparations may be dried
and reused over a series of experiments to give consist
ently high yields of desired steroid ‘and to obtain a high
over and over again.
degree of standardization.
In the usual growing cell fermentations for steriod de
In carrying out the preparation of the dried thalli, a 40 hydrogenation, serious side reactions usually take place
1,2—dehydrogenating organism is grown under normal
which have been designated as “destructase“ activity.
fermentation conditions, and harvested after good growth
With most steroids, destru-ctase activity is widespread and
is achieved, usually within a period of 8 to 72 hours de
results in a greatly decreased yield of the desired 1,2
pending upon the organism and growing conditions. The
dehydrogenated product, and with certain steroids de
thalli are then separated from the supernatant beer by
struction is so rapid and complete that no desired steroid
can be isolated. Destructase activity is manifested by the
simultaneous loss of 240 mp. absorption (due to the
A4—3-ketone), and loss of blue tetrazolium reaction (due
to the 20,2la-ketol group), in those steroids which con
tain these groups. Thus the destructase system, at the
?ltration or centrifugation and may or may not be washed
to remove extraneous material. The liquid remaining is
removed by freeze dry lyophilization or mixing the thalli
with acetone, removing the acetone and air drying. The
dried thalli which is in the form of a powder can be
stored at room temperature or preferably at about 4° C.
very least, simultaneously attacks the “A" ring and “side
chain,” and the reactions involved may be considerably
more profound. Growing cells of many 1,2-dehydro
genating organisms have been found to have a very
active destructase system. It is unexpected and also
unpredictable that the new method of utilizing dried thalli
as described hereinbefore materially reduces the destruc
These preparations of thalli have been shown to be active
for more than a year and may retain their activity in
de?nitely.
In preparing the dried thalli of the present invention,
We can use microorganisms of the class Schizomycetes,
order Eubacteriales, family Corynebacteriaceae, genus
Corynebacterium such as Corynebacteriu'm simplex. Also
microorganisms of the family Mycobacteriaceae such
as Mycobactcrium phlei and of the family Actinomy
60
cetaceae such as Nocardia aurantia and Nocardia coral
lina. We can use members of the Bacteriaceae family,
use of the new process significantly reduces the side reac
such as Bacterium cyclooxydans and microorganisms of
the order Pscudomonadales, family Pseudomonadaceae
such as Pseudomonas testosteroni, members of the Asco
rnycetes class such as Didymella lycopersici and Fungi
Imperfecti such as Septomyxa a?inis can also be used.
tase activity or entirely removes such activity. Because
of this fact, greatly increased yields of a wide variety
of l,2~dehydro steroids have been obtained with the
organisms described hereinafter in the examples. The
tions occurring in the usual fermentations of steriods to
05
produce 1,2-dehydrogenation of such steroids.
The dried thalli preparation of the present invention
can be prepared, for example, by taking 17 hour old cells
of Nocardia corallina (ATCC 999) grown in a medium
The dried thalli are used to carry out a 1,2-dehy
consisting of 0.25% sodium chloride, 0.4% peptone, 1%
drogenation of steroids by mixing with water or a buffer c . ._ glucose, 0.4% beef extract, 0.1% yeast extract with pH
solution such as, for example, 0.05 M Tris [tris-(hy
adjusted to 7.0—7.2 before autoclaving, centrifugating
droxylmethyl-aminomethane] pH 7.6, 0.05 M phosphate
clear of supernatant beer, washing once with water, cen
3,087,864
3
4
trifugating again and mixing the packed thalli with a
tivity demonstrate enhanced activity in the presence of
phenazine methosulfate. When using acetone-dried or
small quantity of water to make a creamy paste. This
paste is added dropwise to 10 volumes of cold acetone
lyophilized thalli of N. corallz'na prepared as described in
producing a ?occulent precipitate. After filtering, the
the procedure above, to which 100 rig/ml. of phenazine
acetone prepared thalli are spread in a thin layer and
methosulfate is added, the following results are obtained:
air dried at room temperature. Alternatively, lyophi
lized thalli of Nocardi coral/incl can be prepared by,
Acetone Dried
Lyophilized
Tlinlli (1 mg]
Th nlli (10 mg]
for example, a 6% 12 hour old inoculum of Nocardia
ml.)
ml.)
corallina (ATCC 999) added to a 200 gallon stainless
Hrs
Hrs
steel tank containing 400 liters of medium as described 10
“gt/100 pg. stelag/100 n2. ste
above. The inoculated medium is then agitated at about
roid
roid
130 r.p.m. Air is supplied at the rate of about 1 vol
ume/volume/minute. It is also preferable to add Hodag
A1 F
F
A1 F
F
oil as an anti-foaming agent in amounts to control the
amount of foam. After 22 hours’ growth at about
2 _______________ . _
(i7
23
99
14
5 _______________ _ _
S0
13
100
10
36° C. the mash is centrifugated in a Sharpless centrifuge.
24 ______________ . _
87
4
98
10
The resulting thalli paste is washed twice with 6 liters of
0.05 1M Tris buffer pH 7.6. After a second wash the
Example III
thalli are suspended in 8 liters of Tris buffer and lyo
philized.
A reaction mixture is prepared consisting of 10 mg.
20
Among the steroids found useful in the process of the
lyophilized N. corallina thalli, 100 pg. phenazine metho
present invention are the following: hydrocortisone,
sulfate, 200 ,ug. hydrocortisone diluted to 1 ml. with 0.05
testosterone, 19-nortestosterone and other cyclopentano
M Tris butfer pH 7.6 and after one hour the reaction
polyhydrophenanthrenes such as described hereinafter in
mixture is centrifuged and the supernatant decanted. The
the examples.
In using the dried thalli for the 1,2-dehydrogenation of
steroids, the following procedure has been found desir
able.
packed thalli are resuspended in fresh buffer containing
steroid and phenazine methosulfate as above. The super
natant is extracted and assayed for content of prednis
A series of test tubes containing 10 ml. of reaction
olone (All?) and hydrocortisone (F). This procedure is
mixture are shaken at 20° C. on a reciprocating shaker.
repeated using the same thalli seven times with the follow
The reaction mixture consisting of 10 ing/ml. of dry
thalli, and 200 lug/ml. of steroid added in from 0.1 ml.
ing results:
to 0.2 ml. of methanol diluted to 10 ml. with 0.05 M Tris
buffer, pH 7.6.
[.Lg./1OD pg. steroid
Aliquots of the reaction mixture con
Run
Percent
A1 F
taining the 200 pg. of steroid are removed at intervals for
assay purposes. An 8-fold volume of water-saturated
A1 F
ethyl acetate is employed for extraction of the steroid.
Aliquots of the solvent extract are used for colorimetric
and paper chromatographic assay.
The following examples describe in detail the use of
the dried thalli and the results obtained with representa
tive steroids of the present invention.
1 _____________________________________ _ _
_
Example I
A reaction mixture (1) consisting of 10 mg./ml. lyo
F
83
4
95
__________________________ _ -
55
7
89
_______________________ _ _
57
33
63
______________________________ _ _
45
45
50
______________________________ . _
30
75
32
6
______________________________ _ _
7 _____________________________________ _ .
32
8t]
83
(53
28
58
All dehydrogenations recorded above occur after one
hour of exposure of hydrocortisone to the reaction mix
ture except run 7 in which steroid is exposed to the re
philized thalli of N. corallina (ATCC 999) and 200
rig/ml. of hydrocortisone (F) diluted to 10 ml. with 0.05
M tris-(hydroxylmethyl)-aminon1etl1ane buffer pH 7.6 is
prepared. A second reaction mixture (Ii) consisting of
10 ml. of 17 hour old N. corallina (ATCC 999) growing
the reaction beyond one hour permits complete dehy
shaker tubes on a reciprocating shaker at 28° C.
thalli reactions permit signi?cantly higher concentrations
action mixture for 16 hours. It is shown that extending
drogenation thru run 4 at least.
Example IV
in Medium #13 (0.25% sodium chloride, 0.4% peptone, 50
The
concentration
of
steroid useable in growing cell
1% glucose, 0.4% beef extract, 0.1% yeast extract) is
dehydrogenations rarely exceeds 200—250 pg./ml., the
prepared to which 200 ,ug./ml. of hydrocortisone (F) is
versatility and control which is readily afforded by dried
added. Both reaction mixtures are placed in 100 ml.
Ali
are removed periodically, extracted with 8 volumes of
to be used. Using the general procedure described here
inbefore for lyophilized thalli of N. corallina, the follow
ethyl acetate and assayed.
ing observations are made:
quots of reaction mixture containing 100 ,ug. of steroid
The following results are obtained:
For ml. of reaction mixture
I
II
pig/100 pg. steroid
[LgJlOO 1.1g. steroid
68
110
116
120
F
a 1 F
54
8
0
0
(‘:5
89
4T
7
100 pg. steroid
Percent
A‘F
Hours
A1 F
24 hour assay, pg!
60
F
‘
PMS
Hydro
’I‘halli (mg)
()ig.)
cortisone
AIF
F
(MM
27
3
0
0
65
100 ................ ..
Example I]
It was found that acetone dried thalli showed the pres
ence of a low order 1,2-dehydrogenase activity. Inves
10 _________________ __
1011
100
70
tigation has shown that addition of a suitable electron
acceptor such as phenazine methosulfate (iPMS) restores
full activity to such a preparation. Furthermore, lyo
philized thalli preparations which show considerable ac 75
100 ________________ __
1, 000
100
95
26
79
200
500
100
104
12
1
90
99
1, (lot)
2, 000
100
92
3
29
97
76
500
103
3
97
1,000
2, GOD
5. 000
10, 000
07
78
88
45
0
0
50
93
100
100
64
33
500
96
34
69
1, 000
2, 000
5, 000
10, 000
96
76
113
8t)
15
4
58
7t)
87
95
66
61
3,087,864
5
6
Example V
Using lyophilized thalli of N. corallina in the general
prises contacting the corresponding 1,2-hydrogenated
procedure described hereinbefore, excellent yields are ob
tained of the 1,2-dehydro derivatives when using the fol
lowing steroid substrates: 4-androsten-3,20-dione; 4
pregnene-3,20-dione; 17 a,21 - dihydroxy-4-pregnene-3,20—
part of dry weight of thalli to two parts of dried weight
of steroid, said dried thalli having been prepared from
a microorganism which 1,2-dehydrogenates steroids and
which is selected from the group consisting of the classes
steroid with an aqueous suspension of dried thalli substan
tially free of spores in a proportion of not less than one
dione; 115,21-dihydroxy - 4 - pregnene-3,20-dione; Qa-?u
Schizomycetes, Phycomycetes, Ascomycetes, Basidiomy
oro-l 1,3,2l-dihydroxy - 4-pregnene~3,20-dione; 170:,2l-dl
cetes and Fungi Imperfecti.
hydroxy-4-pregnene - 3,11,20 - trione; ll?,l7a,2l-trihy
2. The process of claim 1 in which the microorganism
is of the order Pscudomonadales and is Pseudomonas
droxy-4-pregnene - 3,20-dione; 9a-?uoro - 1l?,17a,2l-tri
tcstosteroni.
hydroxy-4-pregncne-3,ZO-dione; 1l/3,16a,l7a,2l - tetrahy
3. The process of claim 1 in which the microorganism
droxy-4-pregnene-3,ZO-dione; 1l?3,17a,21 - trihydroXy-Zm
is of the order Eubacteriales and is Bacterium cyclooxy
methyl-4-pregnene - 3,20-dione; 9a‘?uOt‘O-6B,1l?,l7cz,21 15 daus.
tetrahydroxy-4-pregnene-3,20 - dione; 9a-?uoro-lli9,l6a,
4. The process of claim 1 wherein the microorganism
17a,21-tetrahydroxy-4-pregnene-3,ZO-dione and 9e-?uoro
is of the order Actinomycetales and is Nocardz'a corallina.
113,21-dihydroxy - 16¢,l7a - isopropylidenedioxy-4-preg
5. The process of claim 1 in which the microorganism
10
droxy-4-pregnene - 3,20-dione; 2e,?uoro-l l,8,17<x,21-trihy
nene-3,20-dione.
is of the family Bacteriaccae and is Bacterium mycoides.
6. The process of claim 1 in which the microorganism
is of the family Corynebacteriaceae and is Corynebacte
rium simplex.
7. The process of claim 1 in which the microorganism
is of the family Actinomycetaccae and is Nocardia aurau
Example VI
The following LZ-dehydrogenating organisms, Bac
terium cyciooxydans, Bacterium mycoides, Nocardia
aurantia, Corynebacterium simplex, Mycobacterium phlei,
Pseudomonas testosteroni, Didymella lycopersici and
Septomyxa a?im‘s are grown, washed and lyophilized in 25 tia.
8, The process of claim 1 in which the microorganism
a manner similar to that described for Nocardia corallina.
islof the family Mycobacteriaceae and is Mycobaczerium
The lyophilized thalli preparations are then used to de
hydrogenate hydrocortisone to prednisolone. All prepa
rations carried out this reaction in high yields and showed
,1; Her‘.
that observed with growing cell fermentations.
Example VII
which comprises contacting the corresponding 1,2-hy
9. An improved process for the preparation of 1,2-de
a signi?cant reduction in destructase activity relative to 30 hydrogenated 3-substituted steroids of the pregnane series
drogenatcd steroid with an aqueous suspension of dried,
powdered thalli substantially free of spores in a propor
tion of not less than one part of dried weight of thalli
In addition to an active 1,2-dehydrogenase, lyophiiized
Nocardia corallina thalli showed other enzymatic activi 35 to two parts of dried weight of said steroid, said dried,
powdered thalli having been prepared from a microor
ganism which 1,2-dehydrogenates steroids and which is
esterase activity.
selected from the genus Nocardia.
Using the protocol consisting of per m1.:10 mg. lyoph
10. An improved process for the preparation of 1,2
ilized Nocardia corallina thalli 200 [cg/ml. steroid
diluted to 1 ml. with 0.05 M Tris buffer pH 7.6, reac 40 dehydrogcnated steroids having 18 to 21 carbon atoms
and substituted in the 3‘position which comprises con—
tion is carried out with testosterone, 19-nortestosterone
ties including l7-hydroxy oxidase (l7-keto reductase) and
and 2a-hydroxytestosterone-2a,1i'?-diacetate.
tacting the corresponding 1,2-hydrogenated steroid with
The fol
an aqueous suspension of dried, powdered thalli substan
lowing results are obtained:
tially free of spores in a proportion of not less than one
Testosterone,
Hrs.
Testos-
nl-Testos-
terone,
terone,
percent
percent
Andm_
part of dried weight of thalli to two parts of dried weight
nl-Andro
of said steroid, said dried, powdered thalli having been
prepared from a microorganism which 1,2-dehydrogenates
Stenedmnu stenedionc,
percent
percent
steroids and which is selected from the genus Nocardia.
1 ___________________ _-
70
i0
6 ___________________________________________ __
'20
__________ -_
40
60
21 ______________________________________________________ __
ltleNor-
l9-Nortestosterone,
Hrs.
testosterone,
percent
11. An improved process for the preparation of ‘Al-hy
50
100
with an aqueous suspension of dried thalli substantially
free of spores in a proportion of not less than one part of
dried weight of thalli to two parts of dried weight of said
19-Noran
Estradlol,
dropercent stenedione,
85
percent
10
5
Estrone,
percent
hydrocortisone, said dried thalli having been prepared
55 from Nocardia corallina.
12. An improved process for the preparation of estrone
which comprises contacting l9-nortestosteronc with an
__________ .
60
drocortisone which comprises contacting hydrocortisone
40
aqueous suspension of dried, powdered thalli substantially
100
free of spores in a proportion of not less than one part
60 of dry Weight of thalli to two parts of dried l9-nortestos
2a-HydroxytcstosteronB-QQ,l'l’?-diacetate (QaOIIT-CliAC)
Hrs.
2aOHT- ‘ZqOHT,
diAc,
percent
percent
A120HT,
percent
2110K
AD,
percent
terone, said dried thalli having been prepared from No
cardia corallina.
13. An improved process for the preparation of AI-hy
N‘ZOH
AD,
percent
10
50
100
Formation of estradiol and estrone occurs due to aro
65
drocortisone which comprises contacting hydrocortisone
with an aqueous suspension of dried, powdered thalli sub
stantially free of spores in a proportion of not less than
one part of dried, powdered thalli to two parts of dried
weight of steroid, said dried, powdered thalli having been
matization of A1-19-nortestosterone and A1-19-norandro 70 prepared from Nocardia corallina, allowing the fermenta
tion to proceed until a substantial amount of nl-hydrocor
stenedione. Estrone is also formed by oxidation of the
17 ?-hydroxyl of estradiol to a 17 -ketone.
We claim:
tisone is produced and recovering said nl-hydrocortisone
therefrom.
14. A method of preparing 3-substituted l7-keto A‘
1. An improved aerobic process for the preparation of
3,17-disubstituted 1,2-dehydrogenated steroids which com 75 steroids from the corresponding l7-hydroxyl-1,2-dibydro
3,087,864
7
steroids wihch comprises contacting said steroid with an
aqueous suspension of dried, powdered thalli substan
tially free of spores in a proportion of not less than one
part of dried, powdered thalli to two parts of dried weight
of said steroid, said dried, powdered thalli having been
prepared from a l,2-dehydrogenated specie of the genus
Nocardia.
15. A method of preparing N-androstenedione which
comprises contacting testosterone with an aqueous sus
8
than one part of dried weight of thalli to two parts of
dried weight of said steroid ester, said dried thalli having
been prepared from Nocardz'a corallina.
18. An improved process for the hydrolysis of a 3,17
disubstituted acetoxy steroid to the corresponding hy
droxyl steroid which comprises contacting a 3,17-disub
stituted acetoxy steroid with an aqueous suspension of
dried thalli substantially free of spores in a proportion
of not less than one part of dried weight of thalli to two
pension of dried thalli substantially free of spores in a 10 parts of dried weight of said acetoxy steroid, said dried
proportion of not less than one part of dried weight of
thalli having been prepared from Nocardia corallz'na.
thalli to two parts of dried weight of said testosterone,
19. An improved process for oxidizing a l7»hydroxyl
said dried thalli having been prepared from Nocardt'a
steroid containing 18 to 19 carbon atoms to the corre
corallina.
sponding l7-keto steroid which comprises contacting a
16. A method of preparing A1-2-hydroxyandrostenedi
l7-hydroxyl steroid with an aqueous suspension of dried
one which comprises contacting 2ot-hydroxy testosterone
thalli substantially free of spores in a proportion of not
2a,l7v.-diacetate with an aqueous suspension of dried
less than one part of dried weight of thalli to two parts
thalli substantially free of spores in a proportion of not
of dried weight of said l7-hydroxyl steroid, said dried
less than one part of dried weight of thalli to two parts
thalli having been prepared from Nocara'ia corallina.
of dried weight of said 2ot-hydroxy testosterone-2a,l7a
diacetate, said dried thalli having been prepared from
Nocardia corallina.
17. An improved process for the hydrolysis of an ester
group to a hydroxyl radical of a 3,17-disubstituted steroid
ester which comprises contacting a said 3,l7-disuhstituted
steroid ester with an aqueous suspension of dried thalli
substantially free of spores in a proportion of not less
References Cited in the ?le of this patent
UNITED STATES PATENTS
2,880,205
2,958,631
Campbell et al. _______ __ Mar. 31, 1959
Charney _____________ __ Nov. 1, 1960
3,0l6,335
Stoudt ________________ __ Jan. 9, 1962
3,031,379
Knight ______________ __ Apr. 24, 1962
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